At a superficial level, the concept of a centromere is straightforward: the part of a chromosome that provides a favorable chromatin environment for kinetochore assembly and microtubule attachments during cell division. At a molecular level, however, the concept of a centromere is quite murky and involves a complex interaction between genetic and epigenetic factors. While in many organisms centromeres have been mapped to specific genetic loci and many of their protein components identified, fundamental questions about what regulates centromere identity and function remain unanswered. Recent evidence suggests that RNA plays an important role in centromere chromatin. The central goal of this proposed research is to understand the contribution of chromatin-associated RNA to centromere regulation and function. We have chosen to use the model plant species maize for the proposed experiments because of its unique advantages in studying centromere biology and because of the availability of resources and reagents tailored to our needs. In particular, maize is unrivaled for cytological analysis, provides multiple examples of epigenetic centromere deviants, and has the distinction of being the first organism in which chromatin-bound centromere RNA was detected. The following specific aims will make use of a number of complementary experimental approaches, but will rely substantially on our strengths and experience with characterization of diverse RNA populations through deep sequencing. 1) Survey and characterization of the centromere RNAome, 2) Delineation of the rules-of-engagement for RNA in centromere chromatin, and 3) Identification of novel centromere regulatory factors. We anticipate that this work will lead towards progress in several important areas of research connected to human health including causes of aneuploidy and relation to human disease, rational design of minichromosomes for gene therapy, and understanding of complex heritable epigenetic phenomena (based on the knowledge gained from the exemplary and easily tractable system provided by centromeres). PUBLIC HEALTH RELEVANCE: Modern medicine has greatly benefitted from scientific investigation into the molecular details of the cell and the genes that encode each component. What we hope to gain through our proposed research is a clearer understanding of the centromere, a fundamental cellular structure that is essential for accurate and reliable transmission of genes from one generation to the next. )